Given the strict regulations governing the introduction of new drugs, chirality and related issues have garnered increasing attention in both modern pharmaceutical industries and academic research settings worldwide, especially in the realm of medicinal chemistry projects. Considering the crucial chirality-related issues, the necessity to establish suitable methodologies for determining and monitoring enantiomeric composition in either medicinal products or drug candidates becomes apparent. Among these methods, those utilizing the high-performance liquid chromatography (HPLC) technique through the “direct approach” with chiral stationary phase (CSP) currently offer distinct advantages over the others due to their efficiency and reliability. Hundreds of CSPs are now available on the market, with around 20–30 CSPs, or even fewer, being most commonly utilized to address the majority of enantiomer separation challenges across various compound classes. In this chapter, six separate examples concerning the enantioseparation of medicinal chemistry–relevant compounds of varying chemical and physicochemical nature are presented. In this framework, the use of both high-molecular-weight (polysaccharide- and glycopeptide-based) and low-molecular-weight (amino acid-, Cinchona alkaloid- and fully synthetic-based) chiral selectors is described under the most relevant elution regimens for liquid chromatography (i.e., reversed-phase (RP), normal-phase (NP), and polar organic (PO)/polar ionic (PI)modes).